Stem cells, especially human embryonic stem cells, have recently arisen as a promising cell therapeutic in the regenerative medicine and medical industries. In recognition of the economic and industrial use and added value of human embryonic stem cells, the development of technologies and materials for inducing human embryonic stem cells to differentiate into functionally specific cells has emerged as one of the most interesting topics. As embryonic stem cells were first derived from mouse embryos in 1981 by Evans and Kaufman, mouse embryonic stem cells have been used as basic materials for use in the study of embryonic stem cells and the development of differentiation inducing technologies.
Embryonic stem cells possess two characteristic properties: self-renewal—the ability to go through numerous cycles of cell division under ex vivo conditions without differentiation while maintaining a normal nucleus type; and potency—the capacity to differentiate, at least theoretically, into almost all specialized cell types that constitute the body under culture conditions. These surprising properties greatly require technologies and materials for the development and application of human embryonic stem cells. Since 1998, when a breakthrough in human embryonic stem cell research came when Thomson first developed a technique for isolating and growing cells when derived from the inner cell mass of human blastocysts, active research has been conducted to develop technologies targeting human embryonic stem cells, especially technologies for tissue-specific differentiation-inducing technologies. Many research reports disclose successes in the differentiation of human embryonic stem cells into retinal progenitor cells (Lamba D A et al., 2006), nerve cells (Li X J et al., 2006; Zhang S C et al., 2001), hematopoietic cells (Tian X et al., 2005; Kaufman D S et al., 2005; Kaufman D S et al., 2001), cardiomuscular cells (Kehat I et al., 2003; Kehat I et al., 2001), and pancreatic cells (Assady S et al., 2001) under ex vivo culture conditions, and the possibility of using human embryonic stem cells as cell therapeutics was also suggested (Zhang S C et al., 2001).
The bone maintains the homeostasis thereof through bone formation and remodeling. The site at which active bone remodeling takes place is known as a bone remodeling unit (BRU) or bone multicellular unit, which consists of osteoblasts and osteoclasts, which play critical roles in osteogenesis and bone resorption, respectively. A hindrance to cooperation between the two cells in the remodeling process gives rise to various metabolic bone diseases, including osteoporosis. However, none of the therapies developed thus far ensure complete recovery from metabolic bone diseases. Thus, there is still emphasis on the prophylaxis of metabolic bone diseases in the medical field.
Extensive efforts have recently been made to produce osteoblasts using stem cell differentiation inducing techniques and to apply osteoblasts to the enhancement of bone tissue functions and the treatment of bone tissue injuries. As osteoblasts responsible for bone formation are naturally derived from mesenchymal stem cells (Caplan A I, 1991), immense attention has been paid to the use of mesenchymal stem cells in inducing osteogenic differentiation and as cell therapeutics (Halleux C at al., 2001; Jaiswal N et al., 1997; Hashimoto J et al., 2006; Hofmann S et al., 2007; Xin X et al., 2007; Quarto R et al., 2001). The mesenchymal stem cells of adult tissues may be a useful approach to bone regeneration with autogenous bone grafts, but are disadvantageous in that they are very small in number and difficult to collect by bone marrow aspiration. Hence, the collected cells must be proliferated to a necessary population by ex vivo culturing. However, the fact that a limitation on the possible number of cell division cycles exists and that there is a high possibility of inducing cell modification during the large number of cycles of cell division act as a bottleneck for the use of mesenchymal stem cells. It was reported that nine or more passages cause mesenchymal stem cells to experience aging and lose stem cell properties and osteogenetic potency (Bonab M M et al., 2006). On the other hand, early-stage human embryonic stem cells enjoy the advantage of being applicable to the treatment of various diseases not only because they are a means of understanding the mechanism of osteogenic differentiation, but also because a relatively large number of the cells can be supplied thanks to the ability thereof to proliferate through numerous cycles of cell division under ex vivo culture conditions.
Osteogenic supplements, such as ascorbic acid, β-glycerophosphate, and dexamethasone, are reported to be useful in the induction of osteogenic differentiation (Karp J M et al., 2006; Cao T et al., 2005; Bielby R C et al., 2004; Sottile V et al., 2003). Also reported are induction methods for the differentiation of human embryonic stem cells into osteoblasts by co-culturing with cells derived from bone tissues (Ahn S E et al., 2006), and the differentiation of human embryonic stem cell-derived mesenchymal stem cells into osteoblasts (Barberi T et al., 2005; US2005/0282274 A1). The transforming growth factor-beta (TGF-beta) subfamily, including activin, bone morphogenetic protein (BMP), inhibin, and growth/differentiation factor (GDF), is known to play a critical role in the formation and maintenance of bone tissues. Particularly, human embryonic stem cells are potentially induced to differentiate into osteoblasts when cultured in the presence of BMP2 or BMP4. The understanding of the mechanism by which osteoblasts are differentiated from embryonic stem cells and the techniques of inducing the differentiation have not advanced to a level sufficient to develop cell therapeutics having excellent clinical functions. In order to overcome this, it is important to understand factors involved in osteoblast differentiation and their mechanisms. It is also important to find materials controlling the differentiation factors and utilize them in the induction of osteoblast differentiation.
mTOR (mammalian target of rapamycin), a member of the PIKK (phosphoinositide kinase-related kinase) family, is an important downstream mediator in the PI3k/Akt signaling pathway, which is known to play a critical role in controlling the proliferation and differentiation of embryonic stem cells. Rapamycin binds intracellularly to FK506 binding protein-12 (FKBP12) and the rapamycin-FKBP12 complex targets mTOR, inhibiting its kinase activity, which in turn inhibits the phosphorylation and activation of the downstream translational regulators p70S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). By phosphorylation, mTOR activates the downstream translational regulators, thus promoting various intracellular functions including protein synthesis (Harris T E et al., 2003). Rapamycin, known as an immunosuppressive, is reported to have the activity of inducing the differentiation of various cell lines including mesenchymal stem cells into osteoblasts (Ogawa T et al., 1998; Tang L et al., 2002), and to have a therapeutic effect on osteolysis (US2006/0173033 A1). Also, recent findings suggest that phosphatidic acid, a competitor with rapamycin, activates mTOR, increasing the self-renewal of stem cells while 1-butanol and rapamycin, identified as antagonists of mTOR, inhibit mTOR activity, inducing the differentiation of stem cells (WO 2006/027545 A2, A3).
Many studies on human embryonic stem cells are conducted on the basis of results of research on mouse embryonic stem cells, but it is reported that there are differences between human and mouse embryonic stem cells in proliferation and differentiation properties and relevant molecular regulation mechanisms. Intensive and thorough research into the differentiation of human embryonic stem cells, conducted by the present inventors, resulted in the finding that human embryonic stem cells are induced to effectively differentiate into an osteoblastic lineage in a culture medium supplemented with an inhibitor against mTOR, known to play an important role in a cellular signaling pathway, leading to the present invention.